Diflubenzuron-Induced alterations during in vitro development of Tenebrio molitor
pupal integument.

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Archives of Insect Biochemistry and Physiology 5:201-209 (1987)
Diflubenzuron-Induced Alterations During
In Vitro Development of 'Tenebrio molitor
Pupal integument
N. Soltani, A. Quennedey, J.P. Delbecque, and J. Delachambre
D6partment de Biologie Animale, Universite' d 'Annaba, Algkrie (N.
S.}; Laborafoire de Zoologie,
Unit6 associe'e au CNRS No. 674, Universite' de Bourgogne, Dijon, France (A.Q., J.P.D., J.D.}
The effects of diflubenzuron (DFB) in Tenebrio molitor pupae were first
investigated on cuticle secretion induced by 20-hydroxyecdysone in vitro.
The sternal integuments were treated by DFB either 3 days before culture or
during culture. DFB, when applied before culture, did not prevent the molting
hormone from inducing a new cuticle deposition by integument explants i n
vitro. However, this cuticle showed several architectural alterations and a
thickness reduction. When applied during the culture i n the presence of 20hydroxyecdysone, DFB at high dose ( 2 20 pg/ml) was able t o inhibit cuticle
secretion, but lower doses (< 10 pglml) resulted in epicuticle deposition.
These observations confirm i n vivo studies showing antagonistic effects of
DFB and ecdysteroids at the level of epidermal cells.
In another series of experiments, the DFB effects were analyzed without
addition of exogenous molting hormone in vitro. Because it had been
observed in previous studies that pupal epidermal explants of Tenebrio
secrete low but significant amounts of ecdysteroids in the culture medium,
this in vitro secretion was measured by radioimmunoassay after DFB
treatment. It was observed that DFB, when applied either before or during
culture, significantly reduced the hormonal secretion in vitro. This reduction,
observed at the level of epidermal cells, could be homologous with the
diminution of the endogenous ecdysteroid peak previously described after
i n vivo DFB treatment in Tenebrio pupae.
Key words: Coleoptera, DFB, ecdysteroids, cuticle, in vitro culture, ultrastructure
INTRODUCTION
It is now well established that diflubenzuron (DFB), a substituted urea
growth regulator, inhibits cuticle deposition in insects and some other arthro-
Acknowledgments: We thank Professor C. Noirot, Dr. C. Green, and Dr. B. Mauchamp for
helpful suggestions and we are grateful to Dr. A.C. Grosscurt (Philips-Duphar BV) for kindly
supplying the diflubenzuron.
Received December 9,1986; accepted March 17,1987.
Address reprint requests to Dr. J. Delachambre, Laboratoire de Zoologie, Universiti. de
Bourgogne, 6 Bd Gabriel, 21100 Dijon, France.
0 1987 Alan R. Liss, Inc.
202
Soltani et al
pods: several studies, indeed, have shown a reduction in thickness and
architectural alterations of the cuticle secreted after DFB treatment in vivo [l81. Though it is generally admitted that DFB acts at the epidermal level, DFBinduced disturbances on the ecdysteroid levels or on the 20-hydroxyecdysone metabolism have been also described in several insect species [9-111,
thus leading to the suggestion that DFB could inhibit cuticle deposition via
the control of molting hormone secretion. This hypothesis was strengthened
by observations on the centipede Lithobius [lo] and on the beetle Tenebrio [ll]
showing that, after inhibition of cuticle secretion by high DFB doses, injection
of 20-hydroxyecdysone was able to reinduce cuticle secretion.
The present investigation with Tenebrio rnolitor was thus designed to determine in vitro whether there is a relationship between DFB and ecdysteroids,
as suggested by previous in vivo studies [8,11]. For this purpose, two different kinds of experiments were considered: (1)the effects of DFB were determined on cuticle secretion induced by exogenous 20-hydroxyecdysone in
vitro, the epidermal explants being treated by DFB either before or during
culture: (2) taking into account that the pupal integument of Tenebrio is also
able to secrete small but significant amounts of ecdysteroids in vitro 1121, the
effects of DFB were analyzed on this endogenous hormone secretion.
MATERIALS AND METHODS
Insects
Tenebrio molitor pupae were collected from a stock colony and kept at 27°C
and 70% relative humidity. Under these conditions, the interval between
pupal and adult ecdyses was 6.3 rt 0.3 days (mean 2 SEM).
Tissue Culture
Culture conditions for Tenebrio integument were those described in Quennedey et al. [13]. Each individual explant consisted of four abdominal sternites, sterilely excised, cleaned from fat body and muscle, and washed twice
for 5 min in Landureau culture medium [14]. Then sternites were generally
cultured in sterile dishes (Nunclon) from 1to 10 days at 27"C, floating on 1
ml Landureau medium supplemented with 2% fetal calf serum (Gibco) and
1pglml20-hydroxyecdysone (Simes, Milano, Italy). When experiments were
designed for ecdysteroid secretion measurements, neither calf serum nor
exogenous hormone was added.
DFB Treatment
Treatment with DFB (Philips-Duphar BV, The Netherlands) was made
either before or during culture. For treatment before culture, newly ecdysed
pupae were dipped for 3-4 s in a 10 mglml(32 mM) acetone solution of DFB.
Controls were dipped in acetone alone or not treated at all. Three days after
treatment, abdominal sternites from individuals exhibiting complete developmental inhibition, ie, blocked pupae as previously defined 181, were cultured without addition of DFB in the medium. In the series of experiments
performed for ecdysteroid secretion measurements, sternites from newly
Diflubenzuronin Tenebrio
203
ecdysed pupae were explanted either immediately or 3 days after DFB
treatment.
In experiments in which tissues were treated with DFB in vitro, sternites
from newly ecdysed pupae were cultured in medium that contained 5-25 pg
DFB per ml medium (1.6-8 nM). These concentrations were higher than the
theoretical limit of solubility of DFB into water (0.2 pg per ml). However, we
have verified, using high-performance liquid chromatography as previously
described [15], that such doses added into the culture medium with 5 pl
acetone in a reliably sonicated solution produced relatively stable suspensions, even after several days (more than one-third of the initial dose remained in suspension 3 days after the start of experiment). In controls, the
medium contained either 5 pl acetone or none.
Electron Microscopy
Sternal explants cultured for 1-10 days in 20-hydroxyecdysone-supplemented medium were fixed according to Friend and Farquhar [16], dehydrated, and embedded in an Epon-Araldite mixture. Sections stained with
uranyl acetate and lead citrate were examined with a Hitachi HUllE electron
microscope at 75 kV.
Ecdysteroid Measurements
In the experiments in which the explants were cultured without addition
of exogenous hormone, aliquots from the medium after a 5-day incubation
were extracted with 500 pl ethanol, sonicated, and centrifuged at 10,OOOg for
4 min. The supernatants were evaporated under nitrogen and the extracts
were suitably diluted in 0.1 M citrate buffer (pH 6.2) for duplicate ecdysteroid
measurements via RIA [ l q with antibodies having a 2.5-fold-higher affinity
for ecdysone than for 20-hydroxyecdysone.
RESULTS
DFB Effect on Cuticle Secretion
Insect tissues treated with DFB before culture. The cuticle of a blocked pupa
3 days after DFB treatment, ie, at the start of the incubation, consisted mainly
of preecdysial cuticle and a 1-3-pm postecdysial cuticle with few irregular
lamellae, as already described [8].
Controls (Fig. 1)were explants from DFB-treated pupae (Fig. 2), cultured
in the presence of 20-hydroxyecdysone, were able to undergo apolysis (after
approximately 1day in culture) and then to secrete a new cuticle. However,
instead of the typical helicoidal lamellae seen in controls (Fig. 3), the
endocuticle of treated pupae presented many abnormalities: after a 5-day
culture in the presence of 20-hydroxyecdysone, the thickness was reduced
(1-2 pm vs 3-4 pm) and only electron-dense droplets were observed beneath
the epicuticle (Fig. 4). After a 10-day culture, a lamellate endocuticle with
cuticular canals was deposited in controls (Fig. 5), whereas the dense droplets
of treated explants seemed to coalesce into large globular masses between
epicuticle and epidermis (Fig. 6). Moreover, the epidermal thickness was
considerably reduced.
Fig. 1. A control sternite cultured for 5 days in the presence of 20-hydroxyecdysone. Beneath
the apolysed pupal cuticle (PC), a new cuticle has been secreted by the epidermis (E). Scale
bar = 2 pm. ~3,500.
Fig. 2. A sternite from a diflubenzuron (DFBI-blocked pupa (ie, from a pupa 3 days after
dipping, at ecdysis, in a IO-mg/ml acetone solution of DFB), cultured for 5 days in the presence
of 20-hydroxyecdysone. As in the control, a new cuticle (arrow) has been secreted by the
epidermis (E) under the pupal cuticle (PC). Scale bar = 2 pm. x 3,500.
Fig. 3. Enlarged view of the new cuticle secreted by a control epidermis after a 5-day culture
in the presence of 20-hydroxyecdysone. Lamellate endocuticle (ENDO) can be seen under
the epicuticle (EPI). Scale bar = 1 pm. x 20,000.
Diflubenzuron in Tenebrio
205
The cuticle secreted by control explants from acetone-treated pupae was
similar to that of the untreated ones.
Insect tissues treated with DFB during culture. In the case of in vitro DFB
treatment, the ability of 20-hydroxyecdysone to induce new cuticle secretion
seemed to depend on the DFB concentration in the medium. For 5-10-pglml
concentrations, 20-hydroxyecdysone induced only a thin epicuticle within 5
days of culture (Fig. 7). Higher DFB concentrations (20 and 25 pglml)
prevented any cuticle induction by 20-hydroxyecdysone (Fig. 8).
Fig. 7. A sternite from a newly ecdysed pupa cultured for 5 days in a medium containing 10
pg of DFB per ml. A newly secreted epicuticle (arrow) is present under the apolysed pupal
cuticle (PC). Scale bar = 1 pm. x 10,000.
Fig. 8. A sternite from a newly ecdysed pupa cultured for 5 days in a medium containing 20
pg of DFB per ml. The epidermis (E) has been unable to secrete any cuticular component
under the apolysed pupal cuticle (PC). Scale bar = 1 pm. x 10,000.
Fig. 4. A sternite from a DFB-blocked pupa (same treatment as Fig. 2) after 5 days of culture
in the presence of 20-hydroxyecdysone: enlarged view of the new cuticle secreted by the
epidermis. Under the epicuticle (EPI), only amorphous electron-dense droplets (arrows) have
been secreted. Scale bar = 1 pm. x 20,000.
Fig. 5. A control sternite cultured for 10 days in the presence of 20-hydroxyecdysone. Under
epicuticle (arrow), ten endocuticular lamellae (ENDO) have been secreted by the epidermis
(E). Scale bar = 1pm. ~10,000.
Fig. 6. A sternite from a DFB-blocked pupa (same treatment as Fig. 2) cultured for 10 days.
Under the epicuticle (white arrow), large coalescent droplets (black arrows) secreted by the
epidermis (E) are seen. Scale bar = 1 pm. x 10,000.
206
Soltani et ai
DFB Effect on In Vitro Ecdysteroid Secretion
The amounts of ecdysteroids released into the culture medium by the
epidermis were determined by using RIA after 5-day incubations. Of course
in these experiments no exogenous 20-hydroxyecdysone was added at the
start of the culture. Results obtained with DFB treatments (either before or
during culture) were compared to those of acetone-treated and untreated
controls.
In the case of DFB treatment of newly ecdysed pupae before culture, DFB
reduced the amounts of ecdysteroids secreted in the culture medium by
sternites explanted either immediately or 3 days after treatment (Table 1).
Indeed, the titer in DFB-treated experiments was significantly different from
the titer in untreated or acetone-treated experiments (P < .01 in the t-test for
sternites explanted immediately after DFB treatment and P < .001 for sternites explanted after 3 days). Acetone treatments had no significant effect
(P = .2) on ecdysteroid secretion.
When explanted tissues were exposed to DFB in vitro (Table 2), no significant differences were observed between controls, acetone-treated, and 5-15pglml DFB-treated explants (P > .05). However, at higher DFB concentrations (20-25 pglml), the ecdysteroid secretion was significantly reduced (P <
.OOl) .
DISCUSSION
Compensation of DFB Effects by 20-Hydroxyecdysone
The present in vitro study confirms previous in vivo results, observed in
Tenebrio pupae [ll] and in the centipede Lithobius [lo], about the interactions
between DFB, an inhibitor of cuticle synthesis, and molting hormone 20hydroxyecdysone.
When cultured in vitro, epidermal organs can be induced to secrete a new
cuticle by 20-hydroxyecdysone [18-201. In Tenebrio, our in vitro experiments,
using pupal abdominal sternites, demonstrated that such an induction is also
possible in spite of DFB treatment before culture: 20-hydroxyecdysone can
TABLE 1. Ecdysteroid Titers in Culture Medium
After a 5-day Incubation of Sternites Explanted
Either Immediately or 3 Days After Dipping Into
Acetone Solutions of DFB (10 mglml) or Pure
Acetone or not Treated (Controls)*
Treatment
Control
Acetone
DFB
Ecdysteroid titers (pg) from
sternites explanted
Immediately after
3 days after
treatment
treatment
1,740 k 92
1,727 rf: 69
1,360 k 72
2,089 rf: 164
2,151 I11 173
1,163 rf: 62a
*Each value is the mean rf: SEM of five to nine
cultures measured in duplicate by RIA and
expressed as pgisternite. DFB, diflubenzuron.
aExplants from blocked pupae.
Diflubenzuron in Tenebrio
207
TABLE 2. Ecdysteroid Titers in Culture Medium
Supplemented With 5-25 pg DFBlml, After a 5day Incubation of Explants From Newly Ecdysed
Puvaet
Treatment
Titers (pg)*
Control
Acetone
DFB 5 p g
DFB 10 fig
DFB 15 p g
DFB 20 p g
DFB 25 p g
1,942 f 139 a
1,731 k 68 a
1,707 k 227 a
1,929 k 218a
1,542 236 a
785 rt 123 b
928 f 91 b
Qach value is the mean t SEM of five to ten
cultures measured in duplicate by RIA and
expressed as pgisternite.
*Values followed by the same letter are not
significantly different (P > .05) in the leastsignificative difference test, after variance analysis.
thus suppress the inhibition of cuticle synthesis due to DFB. The new cuticular cycle observed in vitro begins with the depositon of a well-formed
epicuticle by the DFB-treated explants. However, the newly induced endocuticle is thinner than in controls and has many structural abnormalities.
Comparable architectural alterations have also been observed in Pieris larvae
[4,21], Thuumetopoeu larvae [5], Luciliu larvae [22], and in Leptinotuvsu embryos
[6] in which dense amorphous droplets, probably proteinaceaous [23], replace the normal fibrillar structure. These abnormalities are generally related
to chitin biosynthesis alterations [2-41. In Tenebrio, it has been demonstrated
by wheat germ agglutin histochemical binding that the cuticle, induced by
20-hydroxyecdysone after DFB treatment in vivo, contains N-acetyl-aminosugars, thus suggesting the presence of incompletely polymerized compounds [ll] Moreover, in this species, adult differentiations (columnar
cuticle, pit-glands, dermal glands) are also inhibited in vivo, suggesting that
DFB also disturbs the switchover from pupal to adult genetic programs,
probably by interfering with differential mitoses and DNA syntheses [8].
When the DFB treatment is applied in vitro, more pronounced alterations
were observed in Tenebrio pupal sternites. It is possible to completely prevent
the cuticle secretion induced by 20-hydroxyecdysone with DFB concentrations greater than or equal to 20 pglml. However, some cuticle synthesis
(essentially epicuticule) is possible at lower DFB doses. Such results agree
well with studies on Chilo [24], which demonstrate an inverse relationship
between the DFB concentration in the culture medium and the thickness of
the newly secreted cuticle. Although DFB concentrations used in this study
were higher than its theoretical limit of solubility into water, dose differences
were observed: this may be due to the fact that DFB could act not only under
its soluble form but also in suspension or contact. Moreover, it has been
shown that such molecules are efficaciously concentrated by tissues [25].
e
Effect of DFB on Ecdysteroid Secretion
In Tenebrio pupae, the large ecdystroid peak [26] does not originate from
the prothoracic glands, which have already degenerated [27], but from sev-
208
Soltani et a1
era1 other tissues, and particularly epidermis as demonstrated in vivo and in
vitro [12,26,28]. As reported earlier [ll], DFB applied to newly ecdysed
Tenebrio pupae inhibits this ecdysteroid increase in vivo. In the present work,
such an inhibition was also observed at the epidermal level in vitro either for
explants from DFB-blocked pupae or for cultures added with high DFB doses.
These experiments suggest that the hormonal inhibition is at least partly due
to a direct effect of DFB on the epidermis in vitro and possibly also in vivo.
Thus, DFB inhibits the secretion of molting hormone, at least in the peculiar
case of Tenebrio pupal-adult development, in the absence of prothoracic
gland, when the hormonal peak is under the control of epidermis, However,
it appears that in other species or even in Tenebrio larvae, when the molting
hormone peak is under the control of prothoracic glands only, the DFB effect
on the epidermis does not affect ecdysteroid titers (unreported data).
In conclusion, DFB and 20-hydroxyecdysone exert antagonistic and at least
partly reversible effects on epidermal cells for the synthesis of cuticle. However, our experiments suggest that DFB also has endocrine consequences on
the secretion of 20-hydroxyecdysone controlled by epidermis. All these results and other recent reports thus strongly suggest that DFB probably has a
complex mode of action, not only at the chitin synthesis level 129-311 but also
at the hormonal and DNA level [8,11,32-541.
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